2-Mercapto-Quinazolinones as Inhibitors of Type II NADH Dehydrogenase and Mycobacterium tuberculosis:Structure-Activity Relationships, Mechanism of Action and Absorption, Distribution, Metabolism, and Excretion Characterization

<i>Mycobacterium tuberculosis</i> (<i>MTb</i>) possesses two nonproton pumping type II NADH dehydrogenase (NDH-2) enzymes which are predicted to be jointly essential for respiratory metabolism. Furthermore, the structure of a closely related bacterial NDH-2 has been reported recently, allowing for the structure-based design of small-molecule inhibitors. Herein, we disclose <i>MTb</i> whole-cell structure–activity relationships (SARs) for a series of 2-mercapto-quinazolinones which target the <i>ndh</i> encoded NDH-2 with nanomolar potencies. The compounds were inactivated by glutathione-dependent adduct formation as well as quinazolinone oxidation in microsomes. Pharmacokinetic studies demonstrated modest bioavailability and compound exposures. Resistance to the compounds in <i>MTb</i> was conferred by promoter mutations in the alternative nonessential NDH-2 encoded by <i>ndhA</i> in <i>MTb</i>. Bioenergetic analyses revealed a decrease in oxygen consumption rates in response to inhibitor in cells in which membrane potential was uncoupled from ATP production, while inverted membrane vesicles showed mercapto-quinazolinone-dependent inhibition of ATP production when NADH was the electron donor to the respiratory chain. Enzyme kinetic studies further demonstrated noncompetitive inhibition, suggesting binding of this scaffold to an allosteric site. In summary, while the initial <i>MTb</i> SAR showed limited improvement in potency, these results, combined with structural information on the bacterial protein, will aid in the future discovery of new and improved NDH-2 inhibitors

A Design Approach for Collaboration Processes: A Multi-Method Design Science Study in Collaboration Engineering

Collaboration Engineering is an approach for the design and deployment of repeatable collaboration processes that can be executed by practitioners without the support of collaboration professionals such as facilitators. A critical challenge in Collaboration Engineering concerns how the design activities have to be executed and which design choices have to be made to create a process design. We report on a four year design science study, in which we developed a design approach for Collaboration Engineering thatincorporates existing process design methods, pattern based design principles, and insights from expert facilitators regarding design challenges and choices. The resulting approach was evaluated and continuously improved in four trials with 37 students. Our findings suggest that this approach is useful to support the design of repeatable collaboration processes. Our study further serves as an example of how a design approach can be developed and improved following a multi-method design science approach.Multi Actor SystemsTechnology, Policy and Managemen

Chemical moieties associated with CYP inhibition.

<p>Chemical moieties associated with CYP inhibition.</p

Trends of control 1 (closed circles, solvent only control, 0% inhibition), control 2 (open circles, reaction in absence of bactosomes, 100% inhibition) and Z’ (grey squares) of representative high-throughput screening runs of <i>T</i>. <i>cruzi</i> CYP51 FLINT (a) and cytochrome c reductase absorbance (b) assays in 384 well format.

<p>Error bars are the standard deviations of 16 replicates for each control and plate.</p

IC₅₀ Determination of a test set of compounds.

<p>^<b>a</b> Coefficient of variation for all IC₅₀’s were lower than 1%</p><p>IC₅₀ Determination of a test set of compounds.</p

Low systemic absorption of topically applied PPAR β/δ antagonists.

<p><b>A</b>. Peak blood concentrations of PPAR β/δ agonist GW501516, and antagonists GSK0660 and compound 3 h, respectively, at 1 h after topical application to skin. Left: Amount of drugs detected in systemic circulation, expressed as fraction of total amount applied, was calculated as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037097#s4" target="_blank">methods</a>. Right: Drug concentration expressed as molar concentration. <b>B</b>. GSK0660 concentration in blood (left) and total amount of circulating drug as fraction of amount applied (right, calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037097#s4" target="_blank">Methods</a>) at the indicated time points after drug application. The horizontal dashed line represents the reported IC50 for GSK0660 acting on PPAR β/δ reported previously (300 nmol/L). Data shown represent average ± s.d. of n = 3 mice per data point.</p

Absence of inflammatory changes induced by PPAR β/δ antagonists in skin after topical application.

<p>(a) C57Bl/6j wild type mice were treated with ointments containing GSK0660 or compound 3 h applied twice daily to shaved dorsal skin for one week. Mice were sacrificed 1 h after the last ointment application and skin tissue processed for H&E based histology and mass spectrometry, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037097#s4" target="_blank">Methods</a>. Data shown represent average ± s.d. of n = 3 mice per data point (left) treated with GSK (blue columns) or compound 3 h (red). Representative histology sections of all treated mice are shown on right. The inset in the middle panel shows a section of GSK0660-treated epidermis showing apoptotic looking cells (marked by red arrow head). Horizontal bar represents 5 µm. (b) Representative H&E sections of C57Bl/6j wild type mice treated for one week with either GSK0660 (top) or GSK3787 (bottom). Red arrow-heads denoting apoptotic looking cells.</p

Prevention of epidermal hyperplasia by transdermal application of selective PPAR β/δ antagonists.

<p>Both the PPAR β/δ agonist GW501516 (GW) and the antagonists GSK0660 (GSK) or compound 3 h were applied topically to the skin, as described in the text. Left: representative H&E-stained paraffin-sections of dorsal skin from PPAR β/δ transgenic mice after treatment with ointments containing the indicated drugs for twenty days, as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037097#s4" target="_blank">Methods</a>. Horizontal bar represents 5 µm. Right: quantification of H&E-based epidermal thickness observed in n = 4 mice per group, performed as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037097#s4" target="_blank">Methods</a>. * p<0.05 in a two-sided independent t-test.</p

Half – life of GSK0660 after topical application to skin.

<p>42 mg of GSK0660 ointment was applied to dorsal skin of C57Bl/6j wild type mice. Mice were sacrificed at the time after drug application indicated in the figure and drug concentration determined by mass spectrometry, as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037097#s4" target="_blank">Methods</a>. Data shown represent average ± s.d. of n = 3 mice per data point.</p

Characterisation of LdNTR2.

<p>(A) Purification of recombinant LdNTR2 from <i>E</i>. <i>coli</i> BL21(DE3)pLysS [pET15b-LdNTR2]. Lane 1, insoluble fraction; lane 2, soluble fraction; lane 3, pooled fractions from Ni²⁺-affinity chromatography; lane 4, soluble protein following removal of histidine tag; and lane 5, pooled fractions from size-exclusion chromatography. MALDI analysis confirmed that the minor bands represent NTR2 degradation products. (B) Gel filtration profile of the LdNTR2. The inset shows a plot of elution volume against the log molecular mass (MW) of a standard protein mixture (black circles). The red circle represents the elution volume of NTR2. (C) Metabolism of nitroheterocyclic compounds by recombinant NTR2. Initial rates of metabolism were measured in assays containing 100 μM nitro-compound, 100 μM NADPH and 500 nM NTR2. Rates of metabolism with NADPH and NADH alone were 0.0124 ± 0.001 and 0.0084 ± 0.0006 μmol min^-1 mg^-1, respectively. Rates represent the mean ± SD of triplicate measurements. (D) Immunoblots of whole cell extracts (equivalent of 5×10⁶ parasites in each lane) from <i>L</i>. <i>donovani</i> mid-log promastigotes (L), metacyclic promastigotes (M) and axenic amastigotes (A) were probed with LdNTR2-specific polyclonal antiserum. Known amounts of purified recombinant LdNTR2 were loaded as standards for the quantification of the cellular levels of NTR2.</p